Vrana, Julie A.

Context.— Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription–polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish). Objective.— To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. Design.— Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. Results.— Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair (κ = 0.23 and κ = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (κ = 0.75). Interobserver reliability was almost perfect for IHC (κ = 0.91) and fair to moderate for RNAish (κ = 0.38–0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by droplet digital polymerase chain reaction (P = .03, Pearson correlation = −0.65) and were more likely to be positive by RNAish (P = .02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. Conclusions.— Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

Abstract Objectives To evaluate SATB2 expression and prognostic implications in a large cohort of thoracic neuroendocrine tumors. Methods Surgical pathology files (1995-2017) and an institutional thymic epithelial tumor database (2010-2020) were searched for resected neuroendocrine tumors. Cases were stained with SATB2 (clone EP281). Percent SATB2-positive tumor cells and expression intensity were scored. Results In the lung, SATB2 was expressed in 5% or more of tumor cells in 29 (74.4%) of 39 small cell carcinomas and 9 (22.5%) of 40 atypical and 26 (40.6%) of 64 typical carcinoid tumors. SATB2 percent tumor cell expression and intensity were higher in small cell carcinomas than in carcinoid tumors (both P < .001, respectively). After adjusting for tumor subtype, SATB2 expression did not correlate with outcome. In the thymus, four (100%) of four atypical carcinoid tumors and one large cell neuroendocrine carcinoma but no small cell carcinoma (n = 2) expressed SATB2 in 5% or more of tumor cells. Conclusions SATB2 (clone EP281) is expressed in a large subset of pulmonary and thymic neuroendocrine tumors and therefore does not appear to be a useful marker to identify the origin of neuroendocrine tumors. Validation studies are needed, specifically including thymic neuroendocrine tumors, as the expression pattern might be different in those tumors.

Abstract In multiple myeloma, the neoplastic plasma cells are either dispersed throughout the red marrow spaces of the axial and proximal appendicular skeleton or concentrated in discrete, well vascularlized plasmacytomas that stimulate osteoclastic bone resorption and erode into cortical bone. Interactions between bone marrow stromal cells (BMSC) and myeloma cells are clearly important in the pathogenesis of myeloma that diffusely infiltrates the bone marrow and where plasmacytomas are in contact with the bone marrow space. However, these interactions cannot be of major relevance at the centers of expanding plasmacytomas since these sites are devoid of osteoclasts, osteoblasts and other BMSC. Histological sections of skeletal plasmacytomas from 22 patients with multiple myeloma were therefore examined after immunostaining with anti-macrophage antibodies. Hematoxylin and eosin stained sections showed sheets of plasma cells interspersed with numerous small blood vessels. Immunostained sections revealed abundant CD68+, CD163+ macrophage infiltrates in all tumors, and scattered collections of CD3+ T lymphocytes. Tumor-associated macrophages (TAM) accounted for 2 to 12 percent of nucleated cells in these plasmacytomas and were evenly distributed through the parenchyma or clustered around CD34+ blood vessels. In general the TAM had dendritic morphology, and each dendrite was in close contact with multiple plasma cells. In a small percentage of cases, there was a subpopulation of TAM strikingly clustered around CD34+ positive blood vessels. However, vascular mimicry was not seen. Abundant murine CD68+ TAM were also found in subcutaneous KAS6/1 and MM1 myeloma xenografts in SCID mice. To determine whether cells of the monocytic lineage might be exploitable as carriers for systemic delivery of therapeutic agents to plasmacytomas, monocyte-derived dendritic cells were infected with oncolytic measles viruses and administered intravenously to SCID mice bearing subcutaneous KAS6/1 xenografts. Immunostaining for human MHC II antigen revealed that the infused DCs could localize to the KAS6/1 tumors where they heterofused with and transferred MV infection to the myeloma cells. This is the first report of TAM in skeletal plasmacytomas of myeloma patients. Further research into the role of TAM in supporting myeloma growth is warranted since they may offer many potential opportunities for the development of novel approaches to myeloma therapy.

Abstract Peripheral T- and NK-cell lymphomas (PTCLs) are fatal in the majority of patients, and conventional chemotherapy is minimally effective. Targeted inhibition of tyrosine kinases (TKs) might be a useful treatment strategy, but TK overexpression by PTCLs has not been well characterized (except ALK). Recently a novel translocation t(5;9) fusing the ITK and SYK genes was reported in a subset of unspecified-type PTCLs, associated with high SYK gene expression and Syk protein positivity. Syk is a non-receptor TK important in immunoreceptor signal transduction, providing proliferation and survival signals in a variety of hematopoietic cells. Syk is absent or expressed at very low levels in most normal post-thymic T cells, where this signal transduction role is fulfilled by ZAP70. Since Syk TK inhibitors are now in clinical trials for other diseases, we sought to determine the incidence of t(5;9) and Syk overexpression in PTCL. Tissue microarrays were constructed using triplicate cores from paraffin-embedded tissue blocks. Cases were studied using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Additional studies were performed using flow cytometry and Western blot. There were 141 PTCLs, including 35 angioimmunoblastic, 66 unspecified, 21 anaplastic large cell, 2 enteropathy-associated, 5 hepatosplenic, 5 extranodal NK/T, and 7 other. No evidence of t(5;9) was identified by FISH in 86 informative cases. Normal T-cell subsets from peripheral blood, spleen, and lymph node were negative for Syk by IHC, flow cytometry, and Western blot. Syk was positive by immunohistochemistry in 133 of 141 (94%) PTCLs. All 8 Syk negative cases were extranodal, cytotoxic PTCLs. Western blot on frozen tissue correlated with IHC results in paraffin. All 4 Syk-positive cases tested showed phosphorylation of tyrosine residues 525/526, known to be required for Syk activation. We conclude that Syk is overexpressed in most PTCLs, including the vast majority of nodal cases. Syk may substitute for ZAP70 in these tumors and provide ongoing proliferation and survival signaling. The overexpression of Syk and phosphorylation of its Y525/526 residues suggest Syk may be a suitable target for pharmacologic TK inhibition as a treatment option for patients with PTCL.

BACKGROUNDThe objective of the current study was to establish a process for validating immunohistochemistry (IHC) protocols for use on the Cellient cell block (CCB) system.METHODSThirty antibodies were initially tested on CCBs using IHC protocols previously validated on formalin‐fixed, paraffin‐embedded tissue (FFPE). Cytology samples were split to generate thrombin cell blocks (TCB) and CCBs. IHC was performed in parallel. Antibody immunoreactivity was scored, and concordance or discordance in immunoreactivity between the TCBs and CCBs for each sample was determined. Criteria for validation of an antibody were defined as concordant staining in expected positive and negative cells, in at least 5 samples each, and concordance in at least 90% of the samples total. Antibodies that failed initial validation were retested after alterations in IHC conditions.RESULTSThirteen of the 30 antibodies (43%) did not meet initial validation criteria. Of those, 8 antibodies (calretinin, clusters of differentiation [CD] 3, CD20, CDX2, cytokeratin 20, estrogen receptor, MOC‐31, and p16) were optimized for CCBs and subsequently validated. Despite several alterations in conditions, 3 antibodies (Ber‐EP4, D2‐40, and paired box gene 8 [PAX8]) were not successfully validated.CONCLUSIONSNearly one‐half of the antibodies tested in the current study failed initial validation using IHC conditions that were established in the study laboratory for FFPE material. Although some antibodies subsequently met validation criteria after optimization of conditions, a few continued to demonstrate inadequate immunoreactivity. These findings emphasize the importance of validating IHC protocols for methanol‐fixed tissue before clinical use and suggest that optimization for alcohol fixation may be needed to obtain adequate immunoreactivity on CCBs. Cancer (Cancer Cytopathol) 2016;124:89‐99. © 2016 American Cancer Society.

Amyloidosis is caused by deposition in tissues of abnormal protein in a characteristic fibrillar form. There are many types of amyloidosis, classified according to the soluble protein precursor from which the amyloid fibrils are derived. Accurate identification of amyloid type is critical in every case since therapy for systemic amyloidosis is type specific. In ∼20–25% cases, however, immunohistochemistry (IHC) fails to prove the amyloid type and further tests are required. Laser microdissection and mass spectrometry (LDMS) is a powerful tool for identifying proteins from formalin-fixed paraffin-embedded tissues. We undertook a blinded comparison of IHC, performed at the UK National Amyloidosis Centre, and LDMS, performed at the Mayo Clinic, in 142 consecutive biopsy specimens from 38 different tissue types. There was 100% concordance between positive IHC and LDMS, and the latter increased diagnostic accuracy from 76% to 94%. LDMS in expert hands is a valuable tool for amyloid diagnosis.

Amyloidosis affecting lymph nodes (LN) may occur in the setting of systemic amyloidosis or as an entity localized to the site of production (peritumoral). Why some LN amyloid remains peritumoral is unknown. We speculated that the composition of amyloid in these two presentations differs. We analyzed the amyloid proteome in LN amyloid samples to identify differences between the systemic and peritumoral subtypes. In immunoglobulin‐derived LN amyloidosis (N = 26), 70% had heavy chain amyloid (AH or mixed AH/AL). True localized LN amyloidosis was rare, with only 2 patients without a monoclonal protein component. Nineteen patients (73%) had typical amyloid syndromes (100% of AL vs 67% of AH/AL, P = 0.02). A trend to improved survival for the AH/AL group in comparison to AL (median 5‐year survival 48 vs. 19 months, P = 0.06) was seen. Mass spectrometric amyloid analysis is a powerful tool for characterizing amyloid and may provide additional prognostic information. Am. J. Hematol. 88:577–580, 2013. © 2013 Wiley Periodicals, Inc.

Abstract We conducted comprehensive gene expression profiling (GEP) of primary pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma (n = 33) and compared the results to GEP of other B- and T-cell lymphomas and normal lymphocytes to identify novel markers and deregulated pathways. MALT has a prominent T-cell signature and a marginal zone/memory B-cell profile. Four novel transcripts were specifically overexpressed in MALT, and 2 of these, MMP7 and SIGLEC6, were validated at the protein level. GEP also revealed distinct molecular subsets in MALT. One subset, characterized by MALT1 translocations, showed overexpression of nuclear factor-κB (NF-KB) pathway genes but also was enriched for chemokine signaling pathways. Another subset showed increased plasma cells and a prominent plasma cell gene signature. By analyzing several genes with very high (“spiked”) expression in individual cases, we identified clusters with different biologic characteristics, such as samples with MALT1 translocations having high expression of MALT1 and RARA, samples with plasmacytic differentiation having high FKBP11 expression, and samples with high RGS13 expression tending to have trisomy 3 and reactive follicles. In conclusion, MALT subgroups with distinct pathologic features defined by distinct groups of deregulated genes were identified. These genes could represent novel diagnostic and therapeutic targets.